EP2275527A1 - Milieu de culture pour la fermentation bactérienne dépourvu de composants animaux - Google Patents

Milieu de culture pour la fermentation bactérienne dépourvu de composants animaux Download PDF

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Publication number
EP2275527A1
EP2275527A1 EP09100362A EP09100362A EP2275527A1 EP 2275527 A1 EP2275527 A1 EP 2275527A1 EP 09100362 A EP09100362 A EP 09100362A EP 09100362 A EP09100362 A EP 09100362A EP 2275527 A1 EP2275527 A1 EP 2275527A1
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culture medium
lactic acid
medium according
bacterial strains
medium
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German (de)
English (en)
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Intrexon Actobiotics NV
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Actogenix NV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

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  • the invention relates generally to the field of animal component-free culture media for lactic acid bacterial strains and the process for obtaining a high yield of said bacteria making use of said medium.
  • Lactic acid bacterial strains such as for example Lactococcus, Lactobacillus or Bifidobacterium species, are being used in the food industry for many years because they are able to convert sugars (including lactose) and other carbohydrates into lactic acid, and are accordingly useful in the preparation of fermented dairy foods such as yogurt and certain cheese varieties.
  • said lactic acid bacterial strains are beneficial to the body's naturally occurring gut flora, an ecology of microbes, and are accordingly recommended by physicians, and, more frequently, by nutritionists, after a course of antibiotics, or as part of the treatment for gut related candidiasis.
  • the bacteria that work well with our bodies may decrease in number, an event that allows harmful competitors to thrive, to the detriment of our health. Claims are made that living food grade bacteria strengthen the immune system to combat allergies, excessive alcohol intake, stress, exposure to toxic substances, and other diseases.
  • recombinant strains of said food grade bacteria are also being used as promising tools in the intestinal delivery of therapeutic molecules.
  • intestinal delivery of viable recombinant hIL-10 producing Lactococcus lactis (Thy12) was found to have a clinical effect in patients suffering from Crohn's disease ( Steidler L. et al., Nat. Biotechnol. 21(7) (2003) 785-789 ), and in the treatment of Acquired Lactase Deficiency, viable lactase producing bacteria, such as Lactobacillus acidophilus, and Lactobacillus bulgaricus are used in a lactase replacement therapy ( US 6,008,027 ).
  • proteinaceous material from animal origin are used such as for example in the M17 medium for Lactic Streptococci (DSMZ Medium 449), the Lactobacillus medium IV (DSMZ Medium 859), the lactobacilli MRS broth (BD Difco - 2288130), and the Lactobacillus selection broth (BD Difco -211331).
  • DSMZ Medium 449 M17 medium for Lactic Streptococci
  • DSMZ Medium 859 the Lactobacillus medium IV
  • BD Difco - 2288130 lactobacilli MRS broth
  • BD Difco -211331 lactobacillus selection broth
  • non-animal derived culture media have been developed in the past, however, these comprise often complex formulations or they have been specifically developed for particular micro-organism such as the streptococci, meningococci and Haemophilus influenza hereinafter.
  • WO94/09115 discloses a culture medium containing no complex animal components and is optimized for the production of Streptococcus pneumoniae and the isolation of pneumococcal capsular polysaccharide which can be used to prepare vaccines.
  • This SYG medium contains Yeast Extract, Soy Peptone, Resazurin, Vitamin K, Hemin, Sodium Bicarbonate, L-cysteine HCl, Glucose, HEPES buffer (pH 7.3 -7.8) and a salt solution containing CaCl 2 , MgSO 4 , K 2 HPO 4 , KH 2 PO 4 , NaHCO 3 and NaCl.
  • US6933137 discloses an animal-free chemically defined culture medium specifically developed for meningococci comprising sodium phosphate, soya peptone, monosodium glutamate, potassium chloride, magnesium sulphate, L-cysteine and glucose and that does not contain NH 4 Cl.
  • US2002/0137140 discloses a complex lactic acid bacteria culture medium comprising 19 amino acids, K 2 SO 4 , KH 2 PO 4 /K 2 HPO 4 , Na-acetate, CaCl 2 , MgCl 2 , FeSO 4 , vitamins, micronutrients and citric acid.
  • EP0983342B1 discloses a medium comprising at least 20% of dry weight of a soy bean derived protein composition for cultivating Haemophilus influenza.
  • the example medium for cultivating Haemophilus influenza comprises glutamic acid, Na 2 HPO 4 .12H 2 O, KCl, NaCl, NH 4 Cl, NaOH, soytone, proteose peptone, yeast extract, glucose NAD, Tris and Hemin.
  • the accordingly optimized media should ideally only contain essential components, including a carbon source, an energy source, a nitrogen source and essential salts; be easily prepared in a reproducible manner; and support robust high-cell density cultures, with a preferably higher yield compared to the conventional media.
  • the medium disclosed in this invention overcomes most of the difficulties of the conventional bacterial culture medium in that it is much more simplified and as such also easier and cheaper for cultivating large amounts of bacteria. Furthermore, we have surprisingly found that eliminating soy peptone and sodium-containing compounds as well as using NH 4 OH as a pH regulator results in a significantly higher yield at the end of the cultivating process.
  • a culture medium for lactic acid bacteria that does not comprise animal derived compounds and minimally comprises a carbon source, a magnesium source, a nitrogen source, a phosphate buffer and an organic acid.
  • the present invention provides a culture medium for lactic acid bacterial strains characterized by:
  • one or more strains beneficial to the human being can be used as the lactic acid bacterial strains. These may be selected from the group consisting of Lactococcus genus, Lactobacillus genus, Bifidobacterium genus, and any variants, mutants and genetically modified bacteria derived thereof.
  • the organic acid in the culture medium for the lactic acid bacterial strains is selected from the group of organic acids, in particular citric acid and ascorbic acid.
  • the culture medium of the present invention contains a base for buffering the pH, selected from the group consisting of NaOH, and NH 4 OH; in particular a non-sodium containing base such as NH 4 OH.
  • the present invention provides a culture medium for culturing lactic acid bacterial strains characterized by:
  • the pH of the culture medium for lactic acid strains is kept constant during culturing of the lactic acid bacterial strains by continuously monitoring and if necessary adjusting the pH by the addition of base selected from NaOH or NH 4 OH; preferably NH 4 OH.
  • a first objective of the invention is to provide an animal component-free culture medium for growing of high numbers of lactic acid bacterial strains to 10 8 cfu/ml or higher, preferably 10 9 cfu/ml or higher, more preferably 10 10 cfu/ml or higher
  • lactic acid bacterial strains discloses gram-positive, microaerophilic or anaerobic bacterial strains that ferment sugars with the production of acids including lactic acid as the predominantly produced acid.
  • the industrially most useful lactic acid bacteria are found among Lactococcus spp. Streptococcus spp., Lactobacillus spp.
  • lactic acid bacterial stains belonging to the group of the strictly anaerobic bacteria, i.e. Bifidobacterium spp. are generally included in the group of lactic acid bacterial strains.
  • any variants, mutants and genetically modified bacteria derived from the Lactococcus spp. Streptococcus spp., Lactobacillus spp. or Bifidobacterium spp are considered to be lactic acid bacterial strains.
  • the present invention also provides the use of the medium as defined herein for culturing recombinant lactic acid bacterial strains comprising nucleic acids or vectors for expressing heterologous proteins, or for example by introducing exogenous sequences into the thyA locus of Lactococcus ( Steidler et al., 2003 Nat .Biotechnol. 21:785-789 ).
  • Said cultured recombinant lactic acid bacterial strains can subsequently by used for isolation of the recombinant expressed proteins (see, e.g., US 5,559,007 ), or for delivery, especially therapeutic delivery, of said proteins to subjects (see, e.g., WO 97/14806 ; WO 01/02570; Steidler et al., 2003 ).
  • the culture media of the present invention further comprise a component that should complement a deficiency acquired by the recombinant lactic acid bacteria in order to comply with the requirements of biological containment in a deliberate release of recombinant bacteria in the environment, such as for example, but not limited to, a deficiency in the thymidilate synthase A gene where upon such deficient lactic acid bacteria can only grow when a supplement of thymidine or thymine is present in the culture medium. Culture media containing such supplements are also within the scope of the present invention.
  • the culture medium for said lactic acid bacterial strains does not comprise animal derived compounds and minimally comprises a carbon source, a phosphate buffer, a nitrogen source, a magnesium source and an organic acid.
  • the culture medium as provided herein is further characterized in that it is substantially free of sodium, i.e. in that, but for the naturally occurring sodium ions present in the nitrogen source, no further sodium ions are added to the culture medium.
  • the high-yield animal component-free culture medium for lactic acid bacterial strains comprises a carbon source, a phosphate buffer, a nitrogen source, a magnesium source and an organic acid, characterized in that the carbon source, phosphate buffer, magnesium source, and organic acid, are free of sodium.
  • any non-animal carbon source can be used in the culture medium of the present invention and include for example sugars, alcohols, fats, and aromatic esters, carotenoids and terpenes. Particular examples include the sugars, glucose; maltose or lactose.
  • the concentration of the carbon source used in the culture medium of the present invention depends on the type of lactic acid bacteria and the selected cultivation conditions. In a particular embodiment the carbon source is glucose monohydrate. In a preferred embodiment, the concentration of glucose monohydrate is between and about 20-75 g/l, more particular about 55 g/l.
  • any phosphate buffer can be used in the culture medium of the present invention, and include for example Na 2 HPO 4 , NaH 2 PO 4 , K 2 HPO 4 , and KH 2 PO 4 .
  • the phosphate buffer is free of sodium and preferably selected from K 2 HPO 4 , KH 2 PO 4 , or the combination of K 2 HPO 4 and KH 2 PO 4 .
  • K 2 HPO 4 is used as a phosphate buffer at a preferred concentration of between and about 1-20 g/l, more preferably about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 g/l, even more preferably in a concentration range of about 2 - 5 g/l.
  • KH 2 PO 4 is used as a phosphate buffer at a preferred concentration of between and about 1-20 g/l, more preferably about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 g/l, even more preferably in a concentration range of about 2 - 5 g/l.
  • K 2 HPO 4 and KH 2 PO 4 are each independently used at a preferred concentration of between and about 1-20 g/l, in particular K 2 HPO 4 is present at a concentration of between and about 1-20 g/l and KH 2 PO 4 at a concentration of between and about 2 - 5 g/l.
  • any non-animal magnesium source can be used in the culture medium of the present invention and typically consist of a magnesium salt such as for example MgCl 2 , and MgSO 4 .
  • the magnesium salt is MgSO 4 .7H 2 O used at a concentration of between and about 0.1- 2 g/l, preferably about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, or about 1.0 g/l, even more preferably in a concentration range of about 0.2 - 0.5 g/l.
  • any non-animal nitrogen source can be used in the culture medium of the present invention and include for example enzymatic digestion or acid hydrolysis of natural products, such as plants or microbial cultures.
  • Particular examples include; enzymatic digests or hydrolysates of plant material like barley, pea, potato, corn and soya; enzymatic digests or hydrolysates of yeast; or enzymatic digests or hydrolysates of protein mixtures including casein.
  • Bacto Yeast Extract is used as a nitrogen source for the culture medium of the present invention.
  • the concentration of the nitrogen source used in the culture medium of the present invention depends on the type of lactic acid bacteria and the selected culturing conditions.
  • the nitrogen source is Bacto Yeast Extract , used at a concentration of between and about 5-30 g/l, preferably about 5, 10 or about 20 g/l .
  • Bacto Yeast Extract as used in the present invention is defined as a water-soluble extract from autolyzed yeast cells suitable for use in cell culture media and microbial fermentation.
  • any organic acid can be used in the present invention, but preferably ascorbic acid or citric acid is used.
  • the organic acid is used at a concentration of between and about 0.1 - 1 g/l, preferably in a concentration range of about 0.4 - 0.6 g/l.
  • the pH of the medium of the present invention is kept at between and about 7.0 and 7.5, more preferably to about 7.0 or about 7.5. Adjustment of the pH is preferably obtained by the addition of a base selected from the group consisting of NaOH, and NH 4 OH; more preferably by a base not comprising sodium such as NH 4 OH.
  • culture medium is defined as a nutrient medium used in a large-scale fermentation culture wherein microbial cells are grown in a fixed volume under specific environmental conditions (e.g. nutrient type, temperature, pressure, aeration, etc) up to a certain density in a tank or fermentor until they are ready to be harvested and processed as a batch.
  • specific environmental conditions e.g. nutrient type, temperature, pressure, aeration, etc
  • a second objective of the invention is to provide a process for culturing lactic acid bacterial strains comprising the use of the culture medium as defined in the present invention.
  • the means to evaluate the glucose concentration during the batch culture as used in the present invention are known to the person skilled in the art and comprise methods based on the reducing properties of glucose, such as for example the dinitrosalicylic acid assay (DNS) or the ferricyanide assay; or enzymatic assays such as for example using hexokinase or glucose oxidase.
  • DMS dinitrosalicylic acid assay
  • ferricyanide assay ferricyanide assay
  • enzymatic assays such as for example using hexokinase or glucose oxidase.
  • time-temperature conditions, pH conditions and aeration conditions during the process for culturing of the bacteria will depend i.e. on the particular type of lactic acid bacterial strain used. Specific examples of such conditions are provided in the following examples.
  • a culturing under standard conditions is run for between and about 8-24 hours, at a rotation speed of between and about 200-1000 rpm at a temperature range of between and about 15-40°C, and at a pH range of between and about 4-8.
  • the pH is kept constant during the batch cultivation process by continuously monitoring and if necessary adjusting the pH by the addition of a base selected from NaOH or NH 4 OH; preferably NH 4 OH.
  • the main purpose of the formulation and manufacturing process development was to develop an optimised, non-expensive simple, high-yield animal component-free culture medium.
  • Table 1 Composition of the M17 medium Component Concentration Trypton 5 g/l Soya pepton 5 g/l Yeast extract 2,5 g/l Meat extract 5,0 g/l MgSO 4 .7H 2 O 0,51 g/l Na 2 glycerophosphate 19 g/l Glucose 50 or 500 g/l
  • soya pepton and yeast extract were used as a nitrogen source. Since the optimized medium should be animal-free, multiple animal-free soya peptones alone and/or in combination with Bacto Yeast Extract (BD) were tested for their applicability as a nitrogen source. The tested soya peptones were:
  • the optimal ratio of Bacto Yeast Extract and soya pepton was determined by the OD values of bacterial cultures in erlenmeyers with different ratios of Bacto Yeast Extract and the different soya peptones. Since Bacto Yeast Extract alone, always scored better compared to any combination of Bacto Yeast Extract with a soya peptone, it was decided to use only Bacto Yeast Extract at a concentration of 20 g/l for the optimized medium.
  • the final animal-free medium contains the components as indicated in table 2.
  • Table 2 Composition of the OALM medium Component Concentration Bacto Yeast Extract (BD) 20 g/l Citric acid monohydrate 0,49 g/l MgSO 4 .7H 2 O 0,51 g/l Glucose 50 g/l K 2 HPO 4 2 g/l
  • thymidine has been added to the medium because in this experiment a Lactococcus lactis bacterium containing a deficient Thymidilate Synthase A gene has been used for fermentation. Such strain can only grow if thymidine or thymine has been added to the medium as a supplement.
  • the currently optimized culture process is a three step process and includes 2 pre-cultures (in 250 ml and 5 L flasks, filled with 100 ml and 4.9 L of pre-culture medium respectively) and a pH-controlled batch culture step in a 270 L bioreactor (working volume 200 L) with OALM medium.
  • IPC in-process controls
  • the composition of the batch culture medium is depicted in table 2.
  • AOLM batch culture medium
  • a solution containing Bacto Yeast Extract, Citric acid monohydrate and MgSO 4 is prepared and steam-sterilized in situ in the fermentor, after which a 50% glucose + K 2 HPO 4 (2 g/l) solution and a 100mM thymidine solution are filtration-sterilized separately and added aseptically.
  • the total medium volume in the fermentor at this point is 175 ⁇ 5L.
  • the sterile culture medium is sampled for bioburden control.
  • the fermentor is subsequently inoculated with the 5 L PC II. Cultivation is performed at 30 °C and a stirrer speed of 200 rpm for approximately 7 to 9 hours.
  • the pH is controlled at 7.0 by addition of sterile 5 M NH 4 OH.
  • the cultivation measurement and control system continuously registers pH, temperature and stirring speed. Samples are taken during the fermentation as IPC for optical density (OD 600 ) and glucose concentration. Samples are taken towards the end of the fermentation step to verify culture purity and concentration (CFU/ml). The end of the fermentation process is defined as the time point at which the glucose concentration drops below 0.5 g/l, after which fermentor cooling is initiated.
  • the IPC for the batch fermentation process consist of:
  • the maximum OD 600 obtained after these 7-8 hours is 21.96 and 27.5 in 2 different batches, respectively as shown in figure 4 .
  • CFU colony forming units

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EP09100362A 2009-07-17 2009-07-17 Milieu de culture pour la fermentation bactérienne dépourvu de composants animaux Ceased EP2275527A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016207744A1 (fr) * 2015-06-26 2016-12-29 Biobridge Healthcare Solutions Pvt. Ltd. Procédé amélioré pour la production et la purification de polysaccharide pneumococcique capsulaire de streptococcus pneumoniae et procédé de production de composition de vaccin conjugué utilisant ledit polysaccharide

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US6008027A (en) 1997-07-17 1999-12-28 Langner; Bruce J. Enteric polymer coated capsule containing dried bacterial culture for supplying lactase
WO2001002570A1 (fr) 1999-07-05 2001-01-11 Vlaams Interuniversitair Instituut Voor Biotechnologie Distribution de peptides en trefle
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WO2008003781A1 (fr) * 2006-07-07 2008-01-10 Alpro Nv Procédé et milieu de conservation de bactéries d'acide lactique dans un état viable

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WO2005091933A2 (fr) * 2004-03-04 2005-10-06 E-L Management Corporation Methode de traitement de la peau au moyen d'extrait de lactobacillus
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016207744A1 (fr) * 2015-06-26 2016-12-29 Biobridge Healthcare Solutions Pvt. Ltd. Procédé amélioré pour la production et la purification de polysaccharide pneumococcique capsulaire de streptococcus pneumoniae et procédé de production de composition de vaccin conjugué utilisant ledit polysaccharide

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